Wv. Broek et al., High-order autocorrelation with imaging fluorescence correlation spectroscopy: Application to IgE on supported planar membranes, J FLUORESC, 9(4), 1999, pp. 313-324
The use of high-order autocorrelation with imaging fluorescence correlation
spectroscopy is described. Fluorescently labeled, antitrinitrophenyl IgE a
ntibodies were specifically bound to substrate-supported planar membranes c
omposed of trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine an
d dipalmitoylphosphatidylcholine. The IgE-coated membranes were illuminated
with a laser beam that was totally internally reflected at the substrate/s
olution interface. The evanescently excited fluorescence arising from the m
embrane-bound IgE was measured with a CCD camera. The images were corrected
for background and for the elliptically Gaussian spatial dependence of the
evanescent excitation intensity. A series of high-order pixel-to-pixel spa
tial fluorescence fluctuation autocorrelation functions was calculated from
the images, The autocorrelation functions generated multiple independent p
arameters which were used to characterize the nonuniform spatial distributi
ons of the membrane-bound IgE. These parameters varied with the IgE density
and also changed significantly when the IgE-coated membranes were further
treated with unlabeled, polyclonal anti-IgE. The high-order autocorrelation
functions calculated from images of planar membranes containing fluorescen
tly labeled lipids rather than bound, labeled IgE demonstrated that the spa
tial nonuniformities were prominent only in the presence of IgE. Images of
fluorescent beads were used to demonstrate the principles and the methods.